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. 2021 Feb;41(2):154-170.
doi: 10.1002/cac2.12130. Epub 2021 Jan 3.

S100A8 promotes epithelial-mesenchymal transition and metastasis under TGF-β/USF2 axis in colorectal cancer

Si Li  1   2 , Jun Zhang  1   2 , Senmi Qian  1   2 , Xuesong Wu  3   2 , Liang Sun  1   2 , Tianyi Ling  3 , Yao Jin  3 , Wenxiao Li  3 , Lichao Sun  4 , Maode Lai  3   2 , Fangying Xu  1   2
Affiliations

S100A8 promotes epithelial-mesenchymal transition and metastasis under TGF-β/USF2 axis in colorectal cancer

Si Li et al. Cancer Commun (Lond). 2021 Feb.

Abstract

Background: The transforming growth factor-β (TGF-β) pathway plays a pivotal role in inducing epithelial-mesenchymal transition (EMT), which is a key step in cancer invasion and metastasis. However, the regulatory mechanism of TGF-β in inducing EMT in colorectal cancer (CRC) has not been fully elucidated. In previous studies, it was found that S100A8 may regulate EMT. This study aimed to clarify the role of S100A8 in TGF-β-induced EMT and explore the underlying mechanism in CRC.

Methods: S100A8 and upstream transcription factor 2 (USF2) expression was detected by immunohistochemistry in 412 CRC tissues. Kaplan-Meier survival analysis was performed. In vitro, Western blot, and migration and invasion assays were performed to investigate the effects of S100A8 and USF2 on TGF-β-induced EMT. Mouse metastasis models were used to determine in vivo metastasis ability. Luciferase reporter and chromatin immunoprecipitation assay were used to explore the role of USF2 on S100A8 transcription.

Results: During TGF-β-induced EMT in CRC cells, S100A8 and the transcription factor USF2 were upregulated. S100A8 promoted cell migration and invasion and EMT. USF2 transcriptionally regulated S100A8 expression by directly binding to its promoter region. Furthermore, TGF-β enhanced the USF2/S100A8 signaling axis of CRC cells whereas extracellular S100A8 inhibited the USF2/S100A8 axis of CRC cells. S100A8 expression in tumor cells was associated with poor overall survival in CRC. USF2 expression was positively related to S100A8 expression in tumor cells but negatively related to S100A8-positive stromal cells.

Conclusions: TGF-β was found to promote EMT and metastasis through the USF2/S100A8 axis in CRC while extracellular S100A8 suppressed the USF2/S100A8 axis. USF2 was identified as an important switch on the intracellular and extracellular S100A8 feedback loop.

Keywords: S100 calcium-binding protein A8; colorectal cancer; epithelial-mesenchymal transition; metastasis; prognosis; transforming growth factor-β; upstream transcription factor 2.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

FIGURE 1
FIGURE 1
S100A8 is associated with cancer progression and poor survival in CRC patients, and promotes migration and invasion in CRC cells. A‐D. Representative IHC images showing S100A8 cytoplasmic staining in tubular adenocarcinoma (A), mucinous adenocarcinoma (B), lymph node metastases (C), and liver metastases (D) in CRC. E. Kaplan‐Meier survival analysis showed that S100A8 is associated with poor survival in CRC. Survival curves were constructed using the Kaplan‐Meier method with the log‐rank test. 2 cases died within 1 month after surgery were excluded. F‐G. S100A8 overexpression enhanced cell migration and invasion in SW480 (F) and HCT8 (G) cells. H‐I. S100A8 knockdown inhibited cell migration and invasion in DLD1 (H) and HT29 (I) cells. J. Representative H&E staining images (left panel) showed the pulmonary metastatic nodules of the mice injected S100A8 overexpressing HCT8 and control cells (pointed by black arrows). The number of pulmonary metastatic nodules counted in H&E staining was summarized in the right panel. Migration and invasion experiments in vitro were repeated three times. OD values are shown as mean±SD and analyzed by Student's t‐test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. IHC: immunohistochemistry. CRC: colorectal cancer
FIGURE 2
FIGURE 2
S100A8 promoted EMT. A‐B. S100A8 overexpression decreased E‐cadherin, increased Vimentin and nuclear Snail, and changed the cells to a spindle‐like morphology in SW480 (A) and HCT8 (B) cells. C‐D. S100A8 knockdown increased E‐cadherin, decreased Vimentin and nuclear Snail, and changed the cells to a rounded morphology in DLD1 (C) and HT29 (D) cells. The WB results were repeated three times. The gray values are shown in mean ± SD and analyzed by Student's t‐test. *, P <0.05; **, P < 0.01; ns: not significant. WB: Western blot
FIGURE 3
FIGURE 3
USF2 enhanced S100A8 promoter activity and regulatory networks in EMT. A. USF2 enhanced S100A8 promoter activity as verified by the luciferase reporter gene assay. The sequence of the S100A8 promoter was −657/−374 bp upstream from the transcriptional start site. B‐C. ChIP experiments showed that USF2 directly bound to the S100A8 promoter. PCR was performed with primers targeting the binding site (Target) and the negative site (control, B). When the sequence of USF2 binding site was mutated, the luciferase reporting activity decreased significantly (C). This assay was performed in triplicate. Luciferase activity is shown as mean ± SD. wt, wild type; mut, mutant type; *, P < 0.05. D. USF2 overexpression decreased E‐cadherin, increased S100A8, Vimentin, and nuclear Snail, and changed the cells to a spindle‐like morphology in SW480 (left) and HCT8 (right) cells. E. USF2 knockdown increased E‐cadherin, decreased S100A8, Vimentin, and nuclear Snail, and changed the cells to a rounded morphology in DLD1 (left) and HT29 (right) cells. The WB results were repeated three times. The gray values are shown in mean ± SD and analyzed by Student's t‐test. *, P <0.05; **, P < 0.01; ns, not significant. WB: Western blot
FIGURE 4
FIGURE 4
USF2 promotes migration and invasion in CRC cells and is associated with cancer progression and poor survival in CRC patients. A. USF2 overexpression enhanced cell migration and invasion in SW480 and HCT8 cells. B. USF2 knockdown inhibited cell migration and invasion in DLD1 and HT29 cells. C. Representative IHC images showed the coexpression of USF2 cytoplasmic staining (right panel) and S100A8 (left panel) in CRC tissues. D. Kaplan‐Meier survival analysis showed that the patients with negative cytoplasmic USF2 expression had longer survival time (left panel; 1 case died within 1 month after surgery were excluded in survival analysis) and the patients with negative cytoplasmic USF2 and S100A8 expression had longer survival time than other patients (right panel; 6 cases could not detect S100A8 orUSF2 expression due to tissue shedding were excluded in survival analysis). Survival curves were constructed using the Kaplan‐Meier method with the log‐rank test. #, the patients with cytoplasmic USF2‐negative or S100A8‐negative. OD values are shown as mean±SD. ***, P <0.001
FIGURE 5
FIGURE 5
Extracellular S100A8 inhibited EMT by decreasing intracellular USF2/S100A8. A‐B. Extracellular S100A8 (2 μg/mL for 48 h) inhibited nuclear USF2 expression (A) and EMT markers expression (B). The inhibition did not occur in USF2‐overexpressed SW480 cells (B). C. IHC results showed that the number of S100A8‐positive stromal cells was negatively related to USF2 expression in tumor cells. D. Patients with S100A8‐positive stromal cells and USF2‐negative tumor cells had the best outcomes. Survival curves were constructed using the Kaplan‐Meier method with the log‐rank test. The WB results were repeated three times. The gray values are shown in mean ± SD and analyzed by Student's t‐test. *, P <0.05; ns, not significant. IHC: immunohistochemistry
FIGURE 6
FIGURE 6
TGF‐β promoted EMT and cell mobility by up‐regulating USF2/S100A8. A‐B. TGF‐β induced EMT and S100A8 expression in SW480 (A) and DLD1 (B) cells. C‐D. TGF‐β did not promote migration and invasion in DLD1 (C) and HT29 (D) cells with S100A8 knockdown. OD values are shown as mean ± SD. E. TGF‐β increased nuclear USF2 expression. F‐G. Compared to the control, TGF‐β did not induce EMT in USF2 knocked‐down DLD1 (F) and HT29 (G) cells. The WB results were repeated three times. The gray values are shown in mean ± SD and analyzed by Student's t‐test. *, P <0.05; **, P <0.01; ***, P <0.001; ns, not significant
FIGURE 7
FIGURE 7
Feedback loop of S100A8 in TGF‐β‐induced EMT. TGF‐β in the tumor stroma regulated S100A8 expression through USF2 to promote EMT. If there were redundant S100A8 produced by stromal cells in the tumor microenvironment, it would inhibit the USF2/S100A8 pathway of tumor cells, and thereby inhibit EMT in tumor cells.
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